PROJECT SUMMARY The development and differentiation of the skin epidermis is an important area of research since altered keratinocyte function is an underlying cause for various skin conditions including inflammatory skin diseases, cancer and wound healing defects. The transcriptional and epigenetic mechanisms that control development and differentiation of keratinocytes is not well understood, particularly in the in vivo context of the skin. Hence our long-term goal is to examine the chromatin state of keratinocytes and identify and characterize the crucial transcription factors that function as important regulators of epidermal differentiation in health and disease. It is well established that the transcription factor ?Np63 plays a critical role in morphogenesis and differentiation of the skin epidermis, particularly during embryogenesis. However, our current knowledge of the governing principles by which ?Np63 interacts with and shapes the chromatin and transcriptional regulatory environment of keratinocytes is quite limited and based on data amassed primarily from keratinocytes grown in cell culture. Furthermore, if and how ?Np63 plays a role in maintaining adult skin homeostasis after the matured epidermis is formed, has not been adequately addressed due to lack of targeted genetic systems. To address these knowledge gaps, we have generated well-defined ?Np63 transgenic and knockout mouse models that allow us to isolate a pure population of basal-enriched ?Np63+ve keratinocytes, and to perform robust, inducible deletion of ?Np63 in adult tissues. We will leverage the power of emerging genomics and epigenomics toolbox and our newly developed mouse models to address two important questions. To examine the pioneering function of ?Np63, in Aim 1, we will perform ATAC-seq and ChIP-seq experiments and compare the chromatin architecture of embryonic basal keratinocytes of ?Np63-null keratinocytes to their wildtype counterparts. We will also identify ?Np63 targets in vivo and integrate epigenomic and transcriptomic datasets to better understand the ?Np63-dependent gene regulatory mechanisms that are important for embryonic epidermal maturation. In Aim 2, we will generate ?Np63 conditional knockouts and examine how loss of ?Np63 affects the cellular and molecular dynamics of the maintenance and homeostasis program of the adult skin epidermis. Furthermore, we will characterize the ?Np63-governed transcriptional control mechanisms and signaling pathways in adult skin and identify the molecular mechanisms of the skin phenotype encumbered by loss of ?Np63. Collectively, these experiments will shed light on fundamental transcriptional mechanisms of gene regulation and specifically identify keratinocyte-specific regulatory networks on a broad and dynamic scale. Long term, such information has clinical and therapeutic implications for human patients who suffer from disfiguring and debilitating skin diseases resulting from defective keratinocyte differentiation, particularly due to dysregulation in the p63 pathway.